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An Analysis of Telomere Length in Sarcoidosis

¹ Division of Molecular and Clinical Gerontology, Department of Molecular and Cellular Biology and ² Division of Clinical Genetics, Department of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Oita, Japan.

Address correspondence to Toyoki Maeda, MD, PhD, 4546, Tsurumihara, Beppu, Oita, 874-0838, Japan. E-mail: maedat{at}beppu.kyushu-u.ac.jp

	Abstract

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We investigated telomere (terminal restriction fragment [TRF]) length in 111 patients with sarcoidosis regarding both the mean TRF length and the telomere length distribution. A significant decrease was observed in the mean TRF length in sarcoidosis patients in comparison to the age-matched controls, whereas a decreased telomere length was only associated with age in men. The mean TRF shortening seemed to be accelerated in men in their 30s and 50s and in women in their 40s and 50s. We also found a significant decrease with age of telomeres with lengths of 9.4–6.6 kb in men and women in their 20s and an increase of telomeres with lengths of 4.4–2.3 kb in men and women in their 20s and in men in their 50s in sarcoidosis patients versus in the controls who were in their 20s and 50s. These findings suggest the occurrence of age-advanced changes in telomere length in patients with sarcoidosis, regardless of the patient age at the onset of sarcoidosis.

Telomeres, the repeated sequences that cap chromosome ends, undergo shortening with each cell division and serve as markers of a cell's replicative history. Inflammation, which enhances blood cell turnover, and oxidative stress, which increases the rate of telomere erosion per replication, together lead to shortening of telomere length (3). Telomeres, as triple-G-containing structures, are highly sensitive to damage by oxidants (4). Oxidative damage tends to be repaired less thoroughly in telomeric DNA than elsewhere in the chromosome, thereby accelerating telomere loss, whereas antioxidants decelerate it (3). Oxidative stress also triggers arteriosclerotic change in the vascular endothelium (5). Recently, arteriosclerotic disorders have been reported to accompany telomere shortening not only in the vascular endothelium, but also in the peripheral leukocytes, thus suggesting that persistent systemic stress commonly affects oxidative stress in the telomere attrition of peripheral blood cells (6). Immune and inflammatory processes of systemic granulomatous lesions in sarcoidosis may therefore be related to the enhancement of systemic oxidative stress and blood cell turnover, thus leading to the telomere attrition of blood cells.

Recently, several studies in animal models have shown that a loss of a few hundred base pairs from short telomeres could play an important role in cellular aging, but that such a loss may not be detectable by the use of a traditional mean terminal restriction fragment (TRF) analysis (7). As a result, in the present study, we assessed the change of telomere length in sarcoidosis using both the traditional mean TRF (mTRF) length analysis and a percentage analysis of different molecular sizes of the telomere length in the peripheral blood cells in order to answer the following questions: (i) Is there a difference in the telomere length between sarcoidosis patients and controls? (ii) How does telomere length change in sarcoidosis, in which the molecular size is known to have an important association with sarcoidosis?

	MATERIALS AND METHODS

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Study Population
DNA samples were taken from the peripheral blood cells of 111 de novo sarcoidosis patients diagnosed in accordance with Japan Sarcoidosis Society Brain Bank criteria; there were 47 men and 64 women participants ranging in age from 20 to 56 years with a mean age 41.67 years (standard error [SE] 1.10). The diagnosis of sarcoidosis was established based on the clinical and radiographic findings and histologically based on the presence of epithelioid cell granuloma. Among the participants, blood specimens were obtained using 10-mL Vacutainer tubes containing EDTA/heparinized syringes at diagnosis before treatment, then participants were stratified into 10-year age groups. The groups were similar with respect to smoking status, family income, level of physical activity, gender makeup, and socioeconomic status. We also enrolled 126 healthy controls (63 men and 63 women, mean age 44.34 years, SE 0.88) matched for age, sex, and lifestyle. The present research was performed following approval by the Conjoint Health Research Ethics Board of Kyushu University.

Telomeric Length Measurement
Telomeric length was measured as previously described (8–10). Briefly, genomic DNA was extracted from the peripheral blood specimens using PureGene DNA Extraction Kits (Gentra Systems, Minneapolis, MN), and the quality was assessed by agarose gel electrophoresis. Aliquots of DNA (1 µg) were used for a conventional Southern blot hybridization analysis, using a 500-bp-long (TTAGGG)n digoxigenin (dig)-labeled probe specific for telomeric repeats. The blotted membranes were incubated with anti-digoxigenin-specific antibody conjugated with alkaline phosphatase. The telomere probe was visualized by CSPD (C₁₈H₂₀ClO₇PNa₂) (Boehringer Mannheim GmbH, Mannheim, Germany). The membrane was then exposed to Fuji XR film with an intensifying screen. The smears of the autoradiogram were captured on an ImageMaster 2D Platinum (GE Healthcare UK Ltd., Little Chalfont, U.K.), and the telomere length was then assessed quantitatively.

TRF Analysis
Hemann and colleagues (7) and Cherif and coworkers (9) showed that a loss of a few hundred base pairs from short telomeres could play an important role in cellular aging, but it may not be detected by a traditional mean TRF analysis. In this study, we therefore, compared the telomere length using a telomere percentage analysis with four intervals of length as defined by a molecular weight standard. This method has previously been used to determine the change in the telomeres in rats (9). In the present study, we used this method to determine the change in the telomere length in humans. In brief, the intensity of photo-stimulated luminescence (PSL) was quantified as follows: Each telomeric sample was divided into grid squares according to the molecular sizes: 23.1 kb > (23.1–9.4) 9.4 kb, 9.4 kb > (9.4–6.6) 6.6 kb, 6.6 kb > (6.6–4.4) 4.4 kb, 4.4 kb > (4.4–2.3) 2 kb. The percentage of PSL in each molecular weight range was measured (%PSL = intensity of a defined region – background x 100/total lane intensity – background). The mean TRF was estimated using the formula: (ODi – background)/(ODi – background/Li) (11), where ODi is the chemiluminescent signal and Li is the length of the TRF fragment at position i.

Statistical Analysis
The normality of the data was examined using the Kolmogorov–Smirnov test, and the homogeneity of variance with the Levene Median test. After both normal distribution and equal variance of the data were confirmed by these tests, the differences in the telomere length, including the mean TRF length and the findings of a telomere percentage analysis of the age and conditions (sarcoidosis patients or age-matched healthy controls), were analyzed using a two-way analysis of variance (ANOVA) followed by all pairwise multiple comparison procedures using Tukey's post hoc test in men and women, respectively. If the data distribution was not normal distribution, logarithmic or square-root transformations were used to normalize the data for fitting two-way ANOVA. We found no significant difference in the correlation between the age and the telomere length in line regression models. The data are expressed as the mean ± SE. The criterion for significance is p <.05. All analyses were carried out using the Sigma Statistical Analysis Software package (Sigma 2.03, 2001; St. Louis, MO).

	RESULTS

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Comparison of the Two Methods in Order To Analyze the Telomere Length
We assessed the change in telomere length using both the traditional mean TRF length and a percentage analysis of the telomere length distribution in several molecular length regions, and both methods were found to be necessary and important for detecting changes in the telomere length in our study. We could generally estimate the mean telomere length and the telomere attrition rate based on a TRF length analysis (Figure 1), and a more detailed telomere length change could be observed by a percentage analysis. For example, we could not find any difference in mean TRF length between the patients and controls in their 20s (Figure 2), whereas a significant decrease in the longer telomeres measuring 23.1–9.4 kb or 9.4–6.6 kb, and an increase in shortest telomeres measuring 2.3–4.4 kb in patients versus controls was observed based on the findings of a percentage analysis (Figure 3). The combination of the two methods helped us to analyze the telomere length change process more precisely.

View larger version (18K): [in this window] [in a new window]   Figure 1. Relationship between the mean terminal restriction fragment (TRF) length and age in sarcoidosis patients and age-matched controls. A, whole population; B, men; C, women. The mean TRF length in sarcoidosis seems to be shorter than that in age-matched controls, whereas the telomere attrition rate seems the same

View larger version (12K): [in this window] [in a new window]   Figure 2. The mean terminal restriction fragment (TRF) length change with age in sarcoidosis patients and the age-matched controls. Data are mean ± standard error (SE). The mean TRF length was decreased in sarcoidosis patients than age-matched controls in men in their 30s (*p =.005) and 50s (**p <.025) and in women in their 40s (***p <.001) and 50s (****p <.05). The numbers in parentheses are the numbers of analyzed participants

View larger version (19K): [in this window] [in a new window]   Figure 3. Comparison of the percentage change of the telomere length in four molecular sizes with age in the sarcoidosis patients and age-matched controls. The data represent the mean ± standard error (SE). Values of p of age-related significant terminal restriction fragment (TRF) differences are shown. Significant telomere percentage differences are as follows: a (p <.05), b (p <.01), c (p <.005), d (p <.01), e (p <.05), f (p <.05), g (p <.01), h (p <.01), i (p <.05). PSL%, percentage of photo-stimulated luminescence

Age-Related Mean TRF Length Changes
The mean TRF length change with age is shown in Figure 2. The telomere length shortened with age in men from their 20s to 50s (p <.001) (Figure 1), especially for those in their 40s and 50s in comparison to those in their 30s (9.25 ± 0.45, 8.67 ± 0.51 vs 11.87 ± 0.68 kb, p <.025, p <.005, respectively) in the controls, and between those in their 20s in comparison to those in their 50s (10.29 ± 0.55 vs 7.01 ± 0.60 kb, p <.001) in sarcoidosis patients. No significant difference in the telomere length with age was observed in women. In addition, the telomere length in sarcoidosis patients was significantly shorter than that in the controls, especially for men in their 30s (9.03 ± 0.71 vs 11.87 ± 0.68 kb, p =.005) and 50s (7.01 ± 0.60 vs 8.67 ± 0.51 kb, p <.025) and for women in their 40s (8.20 ± 0.53 vs 11.65 ± 0.56 kb, p <.001) and 50s (8.16 ± 0.52 vs 9.64 ± 0.49 kb, p <.05).

Difference of Changes in the Telomere Length Regarding Four Different Molecular Sizes Between Sarcoidosis Patients and Controls
Figure 3 shows the percentage of telomere length regarding four different molecular sizes. In men, the percentage of telomeres measuring 9.4–6.6 kb was lower and the percentage in the shortest telomeres measuring 4.4–2.3 kb was higher in the patients than in controls. In women, we only found the lower tendency for long telomeres, whereas the shortest telomeres increased significantly versus the controls. The differences in the telomere length percentage with age regarding four molecular sizes are shown in Figure 3. We found a significant decrease in the telomere length of 23.1–9.4 kb with age in the male patients, especially between those their 20s and those in their 50s (36.08 ± 2.68 vs 24.29 ± 3.0, p <.025), whereas no difference was observed in the male controls. In addition, a mild but a significant increase was found in the patients versus the controls, for men in their 50s (p <.05). In addition, no difference was seen regarding age between the female patients and controls.

A significant decrease was seen in the telomere length measuring 9.4–6.6 kb in the patients versus the controls in their 20s for both men (26.17 ± 1.23 vs 34.76 ± 1.92, p <.005) and women (24.40 ± 2.23 vs 32.35 ± 1.76, p <.01). In addition, a significant decrease was observed for age in controls in their 30s, 40s, and 50s versus those in their 20s (26.89 ± 1.53, 24.79 ± 1.02, and 24.24 ± 1.13 vs 34.76 ± 1.92, p <.05, p <.001, p <.001, respectively), whereas no difference regarding age was observed for the male patients. Regarding women, no age-related change was found in either the controls or the patients.

No difference in the telomere length measuring 6.6–4.4 kb was seen regarding age for men and women. However, regarding the shortest telomere length measuring 2.3–4.4 kb, a significant increase was found in the patients versus the controls, especially for men in their 20s (16.75 ± 1.91 vs 8.51 ± 2.98, p <.025) and 50s (25.20 ± 2.10 vs 19.54 ± 1.76, p <.05) and for women in their 20s (19.64 ± 3.44 vs 9.74 ± 2.72, p <.05). In addition, we also found an increase in the percentage with age in men, especially for controls in their 40s and 50s versus in their 20s (19.90 ± 1.58, 19.54 ± 1.76 vs 8.51 ± 2.98, p <.01, p =.01, respectively) and for patients in their 50s versus in their 20s (25.20 ± 2.10 vs 16.75 ± 1.91, p <.025). No age-related change was observed in women.

	DISCUSSION

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To our knowledge, this is the first study regarding telomere length changes according to age and gender in sarcoidosis patients. We found a highly significant age-related change in the length of telomeres, including the mean TRF length and the percentage telomere length distribution, in sarcoidosis patients in a well-characterized case–control study. We observed the mean TRF length to be significantly shorter in the blood cells of the sarcoidosis patients than in controls in their 30s to 50s. In addition, we also obtained similar results using a percentage analysis of the telomere length. We observed a significant decrease in the longer telomeres (measuring 9.4–6.6 kb) and an increase in the shortest telomeres (measuring 2.3–4.4 kb) in the patients versus the controls in their 20s. However, telomere attrition rate per year showed almost no difference between them. These findings suggest that changes with age thus occur in the telomere length in sarcoidosis patients, especially in younger ones, thus suggesting that it is meaningful to observe the length changes in the telomeres measuring 9.4–6.6 kb and 4.4–2.3 kb between the patients and controls. The role of the aging process regarding telomere length in sarcoidosis, especially in younger patients, suggests the importance of making an earlier diagnosis for sarcoidosis patients.

We also analyzed the gender-related difference of telomere attrition in sarcoidosis. We did not observe any significant difference regarding age in both the mean TRF length and the percentage analysis in women, whereas the telomeres were significantly shortened as age progressed in men, which is consistent with the previous study that estrogen can stimulate telomerase through an estrogen response element existing on the catalytic subunit of the enzyme (12). Similarly, we found a slower telomere attrition rate in women versus men in both the patients and controls, although the difference was not statistically significant, but at least such a tendency was observed.

Accumulating evidence has shown that telomere shortening is accelerated by increased oxidative stress. A shorter blood cell telomere length in people with an age-related disease has been suggested to relate to increased oxidative stress and chronic inflammation in the disease condition (13).

Based on the speculation that chronic inflammation in sarcoidosis causes continuous oxidative stress, the patients may therefore have demonstrated accelerated telomere attrition in their peripheral blood cells. The cause of sarcoidosis has proven extremely elusive. Many observations are consistent with an infectious or contagious etiology (14–17). Increasing evidence has suggested that high angiotensin-converting enzyme (ACE) levels are useful for monitoring the disease activity (18), and the serum ACE was the only serologic marker in sarcoidosis recommended by the World Association of Sarcoidosis and Other Granulomatous Disorders (19). Furthermore, although there has been no report of an inverse correlation between mean TRF length and serum angiotensin II level, so far, the upregulation of the renin–angiotensin system (RAS) may result in the induction of vascular oxidative stress. It is thought that elevated angiotensin II promotes inflammation processes (20) via the induction in some cytokines (21). The serum ACE level at diagnosis did not correlate with the mean TRF length in sarcoidosis patients in this study (data not shown). The ACE level and angiotensin II level throughout the duration of the disease may be related to the mean TRF. Further investigation will therefore be necessary to elucidate the relationship between serum ACE level and telomere attrition.

Sarcoidosis has been reported to be slightly more predominant in woman than in men (22). A previous study found that estrogen induced vascular endothelial growth factor (VEGF) expression (23), and increasing levels of VEGF, have been shown to be closely related with inflammation and oxidative stress; in contrast, estrogen could also stimulate telomerase (12) while reducing oxidative stress (24). We found a significant shortening of telomeres in the female sarcoidosis patients versus the female controls, although no telomere length change with age was observed, thus suggesting that the latter protection function of estrogen is relatively weak in sarcoidosis. Further studies are thus called for to elucidate the different mechanisms of estrogen between female sarcoidosis patients and controls.

	Acknowledgments

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This work was supported, in part, by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan and by a Grant-in-Aid from Chiyoda Mutual Life Foundation.

We thank Ms. Ueda for her valuable technical assistance. We also thank Dr. Brian Quinn for linguistic advice.

The first two authors equally contributed to the preparation of the manuscript.

Tomokazu Suzuki is now with the Kinki Central Hospital, Kurumazaka, Itami-City, Hyogo, Japan.

	Footnotes

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Decision Editor: Huber R. Warner, PhD

Received April 10, 2007

Accepted June 12, 2007

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